We have generated Adfp-deficient mice and demonstrated that the targeted mice are devoid of Adfp mRNA and protein (Fig. and ). The loss of Adfp does not affect body weight or plasma lipid or glucose levels. Nor does it cause any gross morphological or anatomical changes when the mice are fed a regular chow.
Induction of adipocyte differentiation using cultured MEFs revealed no significant difference in the expression pattern of key regulators or markers of adipocyte differentiation during the transition from fibroblast to adipocyte morphology and no difference in total intracellular TG content after differentiation (Fig. ). In addition, Adfp
-deficient mice have the same fat pad mass as their wild-type littermates (Table ), indicating that absence of ADFP does not lead to a significant impairment in adipogenesis in vivo. Furthermore, Adfp−/−
mice display normal lipolysis under basal conditions and after β-adrenergic agonist stimulation (Fig. ). Together, these data support the conclusion that Adfp
is not essential for adipocyte differentiation or lipolysis. Hence, the burst of Adfp
gene transcription during early adipocyte differentiation appears to be the consequence of increasing lipid accumulation and not the driving force of adipocyte differentiation. In this regard, adipophilin (22
) may be a more appropriate name than ADFP for this gene and protein.
The appearance of Adfp
mRNA during adipocyte differentiation precedes that of PLIN mRNA and protein (3
). LDs are initially coated by ADFP during early adipocyte differentiation, being replaced later by PLIN, although Adfp
mRNA persists after the disappearance of ADFP protein (3
). In this regard, Xu et al. recently showed that ADFP is regulated posttranslationally by proteasomes at the onset of PLIN expression during adipocyte differentiation (70
). We and others showed that Plin−/−
mice display upregulated basal lipolysis in vivo and in isolated adipocytes (40
); an increase of ADFP protein was found in Plin−/−
mice, although functionally, it failed to compensate for the loss of PLIN. Interestingly, we observed no reciprocal increase in Plin
gene expression in adipocytes of Adfp−/−
mice. There was also no compensatory increase in mRNA expression of other PAT domain-containing genes (the Tip47 or S3-12 gene) in these animals (Fig. ), although there seemed to be a small compensatory upregulation of Tip47 protein in WAT of Adfp−/−
mice. It is interesting that the mild Tip47 protein upregulation in the WAT of the Adfp−/−
mice is not associated with an increase in its mRNA, indicating that Tip47 may be regulated posttranslationally in Adfp−/−
mice. It is not clear if the presence of Tip47 compensates for the lack of a WAT phenotype in ADFP deficiency, but it is a possibility that warrants further study. Thus, ADFP does not appear essential for the production or normal functioning of adipocytes; alternatively, the presence of other LD proteins may compensate for the loss of ADFP. It is also possible that ADFP serves a subtle and nonessential adipocyte function for which we have not tested in this study.
In contrast to its nonessential function in lipid accumulation in adipocytes, loss of ADFP in the liver downregulates hepatic TG content (by ~60%). This study establishes an important function of ADFP in the liver that appears not to be replaceable by other LD proteins. The apparently nonoverlapping and different effects of PLIN in adipocytes and of ADFP in hepatocytes suggest that LD proteins may serve unique functions in a tissue-specific manner.
From our experiments, ADFP appears to be important in maintenance of hepatic TG homeostasis because the knockout animals store less TG without changes in cholesterol, NEFA, or PL content in the liver. One possible explanation is that the Adfp−/−
liver has an impaired ability to take up FA. FA uptake has been thought to occur through passive diffusion because it is hydrophobic and could potentially pass through plasma membranes with ease. However, recent studies indicate that the transport of FA, particularly long-chain FA, is a regulated process involving translocases and transporters (19
). A study using transiently transfected COS-7 cells revealed the localization of ADFP to the plasma membrane (16
). More recently, Robenek et al. used freeze fracture electron microscopy and localized ADFP and other PAT family proteins to the cytoplasmic face of the plasma membrane in cultured cells treated with acetylated low-density lipoprotein (49
), consistent with a possible role in FFA uptake. However, we found no significant difference in oleic acid uptake in hepatocytes isolated from Adfp−/−
mice versus those isolated from wild-type mice (Fig. ).
We also found that the Adfp−/−
mice have a TG clearance similar to that of the wild-type. In this study, we used intralipid to circumvent the factors associated with intestinal lipid absorption, chylomicron production, and secretion. It is generally believed that about 60% of the infused intralipid is taken up by rat liver (48
), although the reported hepatic clearance rate has ranged from 20% to 88% (for a review, see reference 25
). Given the 60% reduction of hepatic TG in the Adfp−/−
mice and even if we assume that the hepatic contribution to TG clearance is only 20%, we would still expect to see a difference in the TG clearance curve in the Adfp−/−
mice if TG clearance was the cause for the reduced hepatic content. The identical clearance curve between Adfp−/−
and wild-type mice (Fig. ) makes it highly unlikely that reduced TG clearance by the liver is the cause of low hepatic TG content in Adfp−/−
mice. We cannot exclude the unlikely scenario caused by the presence of compensatory upregulation of muscle TG uptake in the face of a reduced hepatic uptake that is not detected by this experiment.
In one report, L-Fabp−/−
(liver-type fatty acid binding protein knockout) mice under prolonged starvation were also reported to exhibit reduced TG content (47
). We note, however, that another group reported a substantially different phenotype for L-Fabp−/−
), with increased hepatic cholesterol, cholesteryl ester, and PL levels thought to be due to a compensatory upregulation of the Scp
-2 gene. In contrast to either report, we did not observe a change in hepatic cholesterol and phospholipid levels under normal feeding conditions; the reduction in hepatic TG was readily observed without prolonged fasting. Moreover, there was no increase in expression of the L-Fabp
gene in Adfp−/−
mice (data not shown). As such, perturbation of intracellular TG homeostasis in Adfp deficiency appears not to be mediated through L-Fabp
Liver TG reduction was also observed with aP2/mac-1 double-knockout mice (38
) and mitochondrial glycerol-3-phosphate acyltransferase-deficient mice (20
), but these mice have complicated alterations in multiple components of metabolism, such as high energy expenditures, changes in plasma lipids, and increased fatty acid β-oxidation, none of which was evident in Adfp−/−
With a substantial reduction of intracellular TG, one would expect that VLDL secretion would be reduced because studies have shown that degradation of apolipoprotein B (apoB), the obligatory constituent apolipoprotein of VLDL, is enhanced when lipid supply is limiting (14
). However, in the analysis of lipid distribution in different intracellular compartments, we discovered that while TG was reduced in cytosol, it was actually increased in the liver microsome fraction of samples from Adfp−/−
mice (Fig. ). Importantly, there was a concomitant increase in the concentration of MTP in Adfp−/−
mice compared to Adfp+/+
mice (see Fig. S4 in the supplemental material). Increased MTP availability may have contributed to the normal folding of apoB, protecting apoB from degradation and ensuring a normal VLDL secretion rate, despite the presence of reduced hepatic TG content in Adfp−/−
hepatocytes (Fig. ). It is known that TG exists in two different intracellular pools in HepG2 cells, a microsomal pool that is coupled to VLDL secretion and a cytosolic pool that is not (68
). For example, ob/ob mice, which have a severely steatotic liver, have downregulated VLDL secretion (32
) probably because of TG deficiency in the microsomal pool. Our analysis suggests that ADFP plays a crucial role in the balance between the cytosolic and microsomal TG pools in the liver.
Intracellular lipid partitioning in the Adfp−/−
liver is quite different from that of the wild-type liver (Fig. ). In the cytosolic fraction, TG, NEFA, and PL were all reduced. Owing to its strong hydrophobicity, cytosolic fat has to be sequestered in LDs which are generally thought to originate from the outer leaflet of the microsomal membranes, with the polar PL on the surface enclosing neutral lipids, such as TG and cholesteryl esters, in the core (45
). Although NEFA can be carried by the cytosolic fatty acid binding proteins, TG and PL must exist in LDs in the cytosol, and their reduction in the Adfp−/−
(in the cytosol) is an indication of reduced numbers (and size) of LDs, which can be inferred from what we have observed with HFD-treated Adfp−/−
mice (Fig. and see below). The reduction of cytosolic NEFA in Adfp−/−
mice could be a reflection of redistribution of NEFA or reduction of NEFA uptake, although the latter is not supported by direct measurement in this study. In the microsomal fraction, both TG and NEFA were increased in the Adfp−/−
liver. We hypothesize that the absence of ADFP somehow hinders LD formation, leading to the accumulation of TG and NEFA in the microsomal compartment where TG is synthesized.
Under normal chow conditions in C57BL/6 mice, LDs are not readily detectable in the liver even under high-power light microscopy. However, ADFP is upregulated and LDs become readily visible in the fatty liver (22
). We used HFD treatment to induce fatty liver and observed the appearance of LDs in the livers of the wild-type mice and the Adfp−/−
mice. The density of LDs was much reduced in Adfp−/−
mice, being 38% per unit area in these mice compared to that (100%) in wild-type animals. Moreover, LDs in Adfp−/−
livers were also significantly smaller than those in wild-type mice (Fig. ), supporting a role for ADFP in LD formation. There is a reduction of hepatic TG but not PL in Adfp−/−
mice. This is similar to the hepatic fat distribution observed with the chow-fed mice (Table ), suggesting that a similar molecular mechanism operates to reduce TG in the HFD-treated Adfp−/−
LDs received much attention recently with the discovery of many proteins physically localized to these intracellular particles (4
). LD proteins are involved in important biological functions such as lipid metabolism, lipid transport, membrane trafficking, molecular chaperoning, and apoptosis. Nonalcoholic fatty liver disease (NAFLD) is a common ailment in developed countries (21
), manifesting histologically as excessive LD accumulation. This study documents that ADFP deficiency confers resistance to fatty liver development in the presence of normal lipid metabolism. It underscores the use of Adfp−/−
mice as a unique model to study the molecular pathogenesis of NAFLD, while highlighting ADFP as a possible drug target for the prevention and treatment of NAFLD.